Use of a bioadhesive composition comprising a polyphenolic protein

ABSTRACT

A non-irritating, non-allergenic and non-toxic bioadhesive composition can be obtained by providing a bioadhesive composition comprising a) a polyphenolic protein derived from byssus-forming mussels b) a polymer comprising carbohydrate groups. The bioadhesive composition does not contain any enzyme or chemical cross-linking agent. Optionally, the composition may contain an oxidising agent and/or a filler protein. Preferably, the composition is provided as a kit of at least two parts, namely the polyphenolic protein and the polymer comprising carbohydrate groups, respectively. The composition is especially suitable as an adhesive in ophthalmic therapy.

[0001] The present invention relates to biodegradable compositionscomprising adhesive, biocompatible polymers and methods used to coversurfaces and to attach structures to eye tissues, such as the cornea.Polyphenolic proteins isolated from mussels, referred to as “musseladhesive protein” or “MAP”, are used in conjunction with polysaccharidesto achieve strong adhesive bonding. The invention also relates to a kitconsisting of a MAP preparation, a preparation of polysaccharides whichpreferably are negatively charged, and optionally an oxidising agentsuch as hydrogen peroxide nitroprusside ions or periodate ions, that isused for preparing a composition for covering and attaching structuresto eye tissue, such as cornea.

BACKGROUND OF THE INVENTION

[0002] The repair of traumatised structures on and in an eye and itsadnexa is often troublesome. The use of sutures in e.g. the cornea iscausing discomfort, deformations, distortions and may impair the visualacuity. Other eye components, such as the iris, lens structures and theretina are difficult or hardly possible to suture or to join with clipsand related aids. Furthermore, sutures and clips do inevitably induceforeign body reactions and scar formation. The positioning of an implantin ocular structures, e.g. a partial-thickness or a penetratingkeratoplasty or a prosthesis in the cornea, requires the use ofelaborated techniques including the use of haptics to keep it retainedin proper position, which may lead to irritations and adverse reactions.Accordingly, current methods for repairing structures on, at, and in aneye are associated with discomfort and the possibility of inducingpermanent damage to the visual acuity. The use of a composition enablingstructures with wet surfaces to be attached in desired position,remaining adherent for a predicted time period without causing anyopacities in optically important components, or any deformation, scarsor unacceptable foreign body reactions would therefore be highlydesirable.

[0003] Polyphenolic proteins, preferentially isolated from mussels, areknown to act as adhesives. Examples of such proteins can be found ine.g. U.S. Pat. No. 4,585,585. Their wide use as adhesives has beenhampered by problems related to the purification and characterisation ofthe adhesive proteins in sufficient amounts. Furthermore, therequirement for biocompatible suitable cross-linkers and other additiveshave limited their use. Chemicals, such as bifunctional conjugatingcompounds, and enzymes are commonly associated with toxic reactions andother biomedical side effects. Additionally, it is difficult toextensively purify enzymes with retained high activity, avoidingdenaturation and adverse effects on cells, tissues or organs.

[0004] Mussel adhesive protein (MAP) is formed in a gland in the foot ofbyssus-forming mussels, such as the common blue mussel (Mytilus edulis).The molecular weight of MAP from Mytilis edulis is about 130.000 Daltonand it has been disclosed to consist of 75-80 closely related repeatedpeptide sequences. The protein is further characterised by its manyepidermal growth factor like repeats. It has an unusual high proportionof hydroxy-containing amino acids such as hydroxyproline, serine,threonine, tyrosin, and the uncommon amino acid 3,4dihydroxy-L-phenylalanine (Dopa) as well as lysine. It may be isolatedeither from natural sources or produced biotechnologically. U.S. Pat.No. 5,015,677 as well as U.S. Pat. No. 4,585,585 disclose that MAP hasvery strong adhesive properties after oxidation and polymerisation, e.g.by the activity of the enzyme tyrosinase, or after treatment withbifunctional reagents. It is very important in biomedical applicationsof an adhesive and coating composition to use bioacceptable andbiodegradable components, which furthermore should not per se or due tocontamination induce any inflammation or toxic reactions. Fillers,including collagens and polysaccharides, have been added to improve themechanical properties in cases when MAP was used to bond tissues andstructures together, further adding to the risk for immunologicalreactions.

[0005] It is also previously known that it is possible to use adhesivecompositions based on MAP for ophthalmic purposes. Robin et al.,Refractive and Corneal Surgery, vol. 5, p. 302-306, and Robin et al.,Arch. Ophthalmol., vol. 106, p. 973-977, both disclose MAP-basedadhesives comprising an enzyme polymiser. U.S. Pat. No. 5,015,677 alsodescribes a MAP-based adhesive containing a cross-linking agent andoptionally a filler substance and a surfactant. Preferred cross-linkingagents according to U.S. Pat. No. 5,015,677 are enzymatic oxidisingagents such as catechol oxidase and tyrosinase, but sometimes alsochemical cross-linking agents such as glutaraldehyde and formaldehyde.Examples of fillers are proteins, such as collagen and albumin, andpolymers comprising carbohydrate moieties, such as chitosan andhyaluronan. U.S. Pat. No. 5,030,230 also relates to a bioadhesivecomprising MAP, mushroom tyrosinase (cross-linker), SDS (sodium dodecylsulfate, a surfactant) and collagen (filler). The bioadhesive is used toadhere a cornea prosthesis to the eye wall.

[0006] A major problem associated with known MAP-based bioadhesivecompositions, despite the superior properties of MAP per se, is thatsome constituents, in particular the presently used cross-linkingagents, can harm and/or irritate living tissue and cause toxic andimmunological reactions. Chemical crosslinking agents, such asglutaraldehyde and formaldehyde, are generally toxic to humans andanimals, and it is highly inappropriate to add such agents to asensitive tissue, such as the eye. Enzymes, such as catechol oxidase andtyrosinase, are proteins, and proteins are generally recognised aspotent allergens, especially in case they originate from a species otherthan the patient. Because of their oxidising and hydrolysing abilities,they can also harm sensitive tissue. Despite these serious drawbacksassociated with the presently used cross-linkers, it has been regardedas necessary to include them in order to obtain sufficient curing of thebioadhesive.

[0007] Accordingly, there is a need for a MAP-based bioadhesivecomposition which overcomes these drawbacks and hence, does not harm orirritate sensitive tissues such as the eye.

SUMMARY OF THE INVENTION

[0008] Now it has surprisingly been found that a non-irritating,non-allergenic and non-toxic composition can be obtained by providing abioadhesive composition comprising a) a polyphenolic protein derivedfrom byssus-forming mussels b) a polymer comprising carbohydrate groups.The bioadhesive composition does not contain any enzyme or chemicalcross-linking agent. Optionally, the composition may contain anoxidising agent and/or a filler protein. Preferably, the composition isprovided as a kit of at least two parts, namely the polyphenolic proteinand the polymer comprising carbohydrate groups, respectively.

[0009] Definitions

[0010] As disclosed herein, the terms “polyphenolic protein”, “musseladhesive protein” or “MAP” relates to a bioadhesive protein derived frombyssus-forming mussels. Examples of such mussels are mussels of thegenera Mytilus, Geukensia, Aulacomya, Phragmatopoma, Dreissenia andBrachiodontes. Suitable proteins have been disclosed in a plurality ofpublications, e.g. U.S. Pat. Nos. 5,015,677, 5,242,808, 4,585,585,5,202,236, 5,149,657, 5,410,023, WO 97/34016, and U.S. Pat. No.5,574,134, Vreeland et al., J. Physiol., 34: 1-8, and Yu et al.,Macromolecules, 31: 4739-4745. They comprise about 30-300 amino acidresidues and essentially consist of tandemly linked peptide unitsoptionally separated by a junction sequence of 0-10 amino acids. Acharacteristic feature of such proteins is a comparatively high amountof positively charged lysine residues, and in particular the unusualamino acid DOPA (L-3,4-dihydroxyphenylalanine). A polyphenolic proteinsuitable for use in the present invention has an amino acid sequence inwhich at least 5% and preferably 6-25% of the amino acid resdues areDOPA. A few examples of typical peptide units are given below. However,it is important to note that the amino acid sequences of these proteinsare variable and that the scope of the present invention is not limitedto the exemplified subsequences below as the skilled person realisesthat bioadhesive polyphenolic proteins from different sources can beregarded as equivalent:

[0011] a) Val-Gly-Gly-DOPA-Gly-DOPA-Gly-Ala-Lys

[0012] b) Ala-Lys-Pro-Ser-Tyr-diHyp-Hyp-Thr-DOPA-Lys

[0013] c) Thr-Gly-DOPA-Gly-Pro-Gly-DOPA-Lys

[0014] d) Ala-Gly-DOPA-Gly-Gly-Leu-Lys

[0015] e) Gly-Pro-DOPA-Val-Pro-Asp-Gly-Pro-Tyr-Asp-Lys

[0016] f) Gly-Lys-Pro-Ser-Pro-DOPA-Asp-Pro-Gly-DOPA-Lys

[0017] g) Gly-DOPA-Lys

[0018] h) Thr-Gly-DOPA-Ser-Ala-Gly-DOPA-Lys

[0019] i) Gln-Thr-Gly-DOPA-Val-Pro-Gly-DOPA-Lys

[0020] j) Gln-Thr-Gly-DOPA-Asp-Pro-Gly-Tyr-Lys

[0021] k) Gln-Thr-Gly-DOPA-Leu-Pro-Gly-DOPA-Lys

[0022] As disclosed herein, the term “polymer comprising carbohydrategroups”, relates to a naturally occurring or synthetic polymercomprising a plurality of carbohydrate groups. The polymers can beconstituted of discrete carbohydrate groups joined by hydrocarbonchains, but preferably the polymers are polysaccharides, and still morepreferably polysaccharides comprising charged groups. Examples ofsuitable polymers are heparin, chondroitin sulfate, chitosan andhyaluronan.

[0023] As disclosed herein, the term “pharmaceutically acceptable finefilaments” relates to thin fibres which can be used in sutures,preferably ophthalmic sutures. For the purposes of the presentinvention, the lengths of the fibres should not exceed 15 mm, and aretypically within the range of 1-10 mm.

[0024] As disclosed herein, the term “filler protein” relates to aprotein that is optionally added to the bioadhesive composition in orderto obtain a bioadhesive composition that is adapted to specialapplications. Suitable filler proteins according to the presentinvention are collagen, casein, albumin, elastin, fibrin andfibronectin.

[0025] By the term “enzymatic oxidising agent” is meant an enzyme havingthe ability of oxidising MAP in order to promote full or partialcross-linking of MAP and/or polymers and/or filler proteins. Examples ofsuch enzymes according to the state of the art include catechol oxidaseand tyrosinase. A composition according to the present invention doesnot include an enzymatic oxidising agent. By the term “non-enzymaticoxidising agent” is meant a pharmaceutically acceptible oxidising agentwhich, at the doses employed, is non-toxic and non-irritating. Examplesof such non-enzymatic oxidising agents are hydrogen peroxide and sodiumnitroprusside.

[0026] By the term “chemical cross-linking agent” is meant a compoundcomprising at least two functional groups that are able to covalentlycouple to MAP and/or polymers and/or filler proteins. Examples of suchcompounds according to the state of the art include glutaraldehyde,formaldehyde, bis(sulfosuccinimidyl) suberate and 3,3′-dithiobis(sulfosuccinimidyl propionate). A composition according to the presentinvention does not include any chemical cross-linking agent.

[0027] A composition according to the present invention comprises twomandatory components, namely a) a bioadhesive polyphenolic protein, andb) a polymer comprising carbohydrate groups. It is preferred that thecomposition is supplied as a kit of parts, wherein the above mentionedmandatory components are comprised in separate preparations. Thesepreparations are mixed immediately before use. In the completecomposition, the following concentrations have been found to be useful:Concentration Component (mg/ml) polyphenolic protein (component a)0.1-50, preferably 0.3-10 polymer with carbohydrate groups 0.1-50,preferably 0.3-30

[0028] Optionally, a non-enzymatic oxidising agent can be included. Onesuch agent is hydrogen peroxide, which typically can be included in anamount of 1-100 mg/ml, preferably about 10 mg/ml corresponding to 1%(w/v). Other such agents are nitroprusside ions and periodate ions.Periodates, such as sodium periodate, are typically in a concentrationof 2 mM counted on the final composition. Furthermore, pharmaceuticallyacceptable fine filaments can be included in an amount of 0.5-40 mg/ml,preferably 1-20 mg/ml of the final composition.

[0029] In case the composition is supplied as a kit of parts, eachcomponent is provided in the same or a higher concentration, but theabove mentioned concentration ranges will be obtained upon mixing thecomponent preparations with each other in order to prepare the finalcomposition.

[0030] The present invention will now be further described in thefollowing non-limiting examples.

EXAMPLE 1

[0031] Extensively purified mussel adhesive protein ( MAP ) was used,supplied by BioPolymer Products AB, Floda, Sweden, in 5% acetic acid ata concentration of 0.9 mg/mL, and stored in the darkness in the cold(˜8° C.). Heparin from swine intestinal mucosa was purchased from SigmaChemical Co., St. Louise, Mo., USA (H 3393). Additional laboratorychemicals were of highest available purity and purchased from Sigma andMerck.

[0032] The pH of the MAP solution was adjusted to a slightly alkalinepH, usually to 7,5-8.5 but in additional experiments up to about 9.5.The experiments were performed on anaesthetised rats.

[0033] The cornea was deepithelialized. A wound was created surgicallyin the centre of the cornea, with the aid of a trephine with diameter of3 mm. The Bowmans membrane and stroma was excised with a knife and finescissors, down to the vicinity of Descemets membrane. A block of cornealtissue was thereby isolated and removed from its original site. At least5 μL of MAP and 3 μL 10 mg/ml aqueous heparin solution was administratedinto the wound cavity and thereafter were the shortly before removedcorneal tissue pieces repositioned into the cavity to test for adhesionand reattachment mediated by the MAP glue. Adherents was achievedbetween the stroma fragments and the wound cavity in the cornea after 5minutes, as very gently tested with the aid of ophthalmic tweezers andan operating microscope. The MAP-glue combination did not add anyobvious extra intensity with regard to the inflammatory reactions duringthe first days. The corneal wound was covered by epithelium within twodays. No adverse effects could be recognised by visual inspection or bymicroscopy that could be related to the MAP composition.

[0034] Histopathological examinations of those corneae, that still hadstromal fragments attached, after 5 and 7 days revealed that theMAP-heparin combination seemingly did not aggravate the inflammatoryresponse in either the cornea or in the limbus and conjunctiva ascompared to that in specimens from animals having had a wound cavity inthe cornea for the same time period. There was in places down-growth ofepithelial cells into the corneal wound beneath the reattached stromalfragments, but not to that extend that they detached.

EXAMPLE 2

[0035] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1%citric or 1% lactic acid had its pH adjusted to usually to 7,5-8.5, butin some experiments up to about 9.5. Heparin and laboratory chemicalswere of highest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

[0036] The cornea was deepithelialized. A wound was created surgicallyin the centre through roughly half of the cornea, with the aid of atrephine with diameter of 3 mm. The Bowmans membrane and stroma wasexcised with a knife and fine scissors, down to the vicinity ofDescemets membrane. A block of corneal tissue was thereby isolated andremoved from its original site. At least 5 μL of MAP, 3 μL 10 mg/mlaqueous heparin solution and 2 μL 6% (w/v) aqueous hydrogen peroxide wasadministrated into the wound cavity, either once or twice. Thereafterwas the shortly before removed corneal tissue pieces repositioned intothe cavity to test for adhesion and reattachment mediated by the MAPglue. Bonding was achieved between the stroma fragments and the woundcavity in the cornea after 5 minutes, as very gently tested with the aidof ophthalmic tweezers and an operating microscope. The MAP-gluecombination did not add any obvious extra intensity with regard to theinflammatory reactions during the first days. The corneal wound wascovered by epithelium within two days. No adverse effects could berecognised by visual inspection or by microscopy that could be relatedto the MAP composition.

[0037] Histopathological examinations of corneae with stromal fragmentsattached, after 5 and 7 days revealed that the MAP-heparin combinationseemingly did not aggravate the inflammatory response in either thecornea or in the limbus and conjunctiva as compared to that in specimensfrom animals having had a wound cavity in the cornea for the same timeperiod. There was in places down-growth of epithelial cells into thecorneal wound beneath the reattached stromal fragments, but not to thatextend that they detached due to loss of contact with the cornealstroma.

EXAMPLE 3

[0038] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1%citric or 1% lactic acid had its pH adjusted to usually to 7,5-8.5, insome experiments up to about 9.5. Chondroitin sulfate and laboratorychemicals were of highest available purity and purchased from Sigma andMerck. The experiments were performed on anaesthetised rats according tothe rules specified by the ethical permissions. The cornea wasdeepithelialized. A wound was created surgically in the centre throughroughly half of the cornea, with the aid of a trephine with diameter of3 mm. The Bowmans membrane and stroma was excised with a knife and finescissors, down to the vicinity of Descemets membrane. A block of cornealtissue was thereby isolated and removed from its original site. At least5 μL of MAP, 3 μL 24 mg/ml aqueous chondroitin sulphate and 2 μL 6%(w/v) aqueous hydrogen peroxide was administrated into the wound cavity,either once or twice. Thereafter was the shortly before removed cornealtissue pieces repositioned into the cavity to test for adhesion andreattachment mediated by the MAP glue. Bonding was achieved between thestroma fragments and the wound cavity in the cornea after 5 minutes, asvery gently tested with the aid of ophthalmic tweezers and an operatingmicroscope. The MAP-glue combination did not add any obvious extraintensity with regard to the inflammatory reactions during the firstdays. The corneal wound was covered by epithelium within two days,although there seemed to be irregularities in the epithelial celllayering. No adverse effects could be recognised by visual inspection orby microscopy that could be related to the MAP composition.

[0039] Histopathological examinations of corneae with stromal fragmentsattached, after 5 and, for one rat, 7 days revealed that the MAP-chondroitin sulphate combination seemingly did not aggravate theinflammatory response in either the cornea or in the limbus andconjunctiva as compared to that in specimens from animals having had awound cavity in the cornea for the same time period. There was nodistinct down-growth of epithelial cells into the corneal wound.

EXAMPLE 4 -

[0040] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1%citric or 1% lactic acid had its pH adjusted to usually to 7,5-8.5, insome experiments up to about 9.5. Hyaluronan (aqueous solution, 10mg/ml), chitosan (aqueous solution, 10 mg/ml) and laboratory chemicalswere of highest available purity. The experiments were performed onanaesthetised rats according to the rules specified by the ethicalpermissions.

[0041] The surgical procedure was performed as in exampel 1-3. Fairlygood bonding were achieved between the stromal pieces and thesurrounding orignal cornea. The achieved results were in agreement withthose reported in Example 1-3. However, histopthalogical examinationrevealed that was some down-groth of peithelial cells along the lateralborders of the implanted stroma, but to a limited extent.

EXAMPLE 5

[0042] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% lacticacid had its pH adjusted to usually to 7,5-8.5, in additionalexperiments occasionally up to about 9.5. Heparin and laboratorychemicals were of highest available purity and purchased from Sigma andMerck. The experiments were performed on anaesthetised rats according tothe rules specified by the ethical permissions.

[0043] The cornea was deepithelialized. The cornea was carefully punchedin the centre with the aid of a trephine with diameter of 3 mm. Thetissue cylinder was slowly retracted to at least half of its thicknessfrom the original cornea with the aid of fine-pointed instruments undermicroscopic observation. A solution, prepared immediately before use, of20 μL MAP, 10 μL 10 mg/ml aqueous solution of heparin and 10 μL 6% (w/v)aqueous hydrogen peroxide was applied along the border between the plugand the remaining cornea. The cornea plug was then repositioned andgently kept in position. Adhesion was achieved between the central plugand peripheral part of the original cornea after 5 minutes, as verygently tested with the aid of ophthalmic tweezers and an operatingmicroscope.

[0044] Visual inspection and microscopy was performed during 5 days, andoccasionally 7 days. There was an inflammatory reaction in theconjunctiva and blood vessels tended to grow towards the area ofsurgery. The central cornea remained in position fixed to thesurrounding cornea. The cornea was re-epethilialized in 2 days. Therewas edema and slight to moderate opacities in and adjacent to the woundarea. Histopathology of the cornea revealed a slight to moderateinflammation along the zone of surgery. Epithelial cells cover theanterior surface but as well could be detected as strings along partsthe lateral interface of the corneal plug. Leukocytes and macrophagesadhered to the zone of injury, facing the anterior chamber. The achievedresults thus indicate that MAP in conjunction with heparin and anoxidising agent could be used to safely glue a corneal plug back to itssite of surgery.

EXAMPLE 6

[0045] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% citricacid had its pH adjusted to usually to 7,5-9.5. Heparin and laboratorychemicals were of highest available purity and purchased from Sigma andMerck. The experiments were performed on anaesthetised rats according tothe rules specified by the ethical permissions.

[0046] The cornea was deepithelialized. Surgery and application of thebonding mixture was performed as in example 5. Adhesion was achievedbetween the central plug and peripheral part of the original corneaafter 5 minutes, as very gently tested with the aid of ophthalmictweezers and an operating microscope.

[0047] The results obtained was in good agreement with those presentedin example 5 for in this case MAP in citric acid and heparin, both withregard to the clinical outcome and the histopatholoy as investigatedafter 5 days.

EXAMPLE 7

[0048] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% citricacid had its pH adjusted to usually to 7,5-8.5. Chondroitin sulphate (24mg/ml aqueous solution) and laboratory chemicals were of highestavailable purity and purchased from Sigma and Merck. The experimentswere performed on anaesthetised rats according to the rules specified bythe ethical permissions.

[0049] The cornea was deepithelialized. Surgery and application of thebonding mixture was performed as in example 5. Adhesion was achievedbetween the central plug and peripheral part of the original corneaafter 5 minutes, as very gently tested with the aid of ophthalmictweezers and an operating microscope.

[0050] Visual inspection and microscopy was performed during 5 days, andoccasionally 7 days. There was an inflammatory reaction in theconjunctiva and blood vessels tended to grow towards the area ofsurgery. The central cornea autograft remained in position fixed to thesurrounding cornea. The cornea was re-epethilialized in 2 days. Therewas edema and slight to moderate opacities in and adjacent to the woundarea. Histopathology of the cornea revealed a slight to moderateinflammation in the zone of surgery. Epithelial cells cover the anteriorsurface. However, along the lateral interface of the plug and the corneaonly short, but seemingly wide papilla of epithelial cells could berecognised. In none of the sections examined reached the epithelium morethan about one fourth or one third of the depth of the corneal stroma.Leukocytes and macrophages adhered to the zone of injury, facing theanterior chamber. The achieved results thus indicate that MAP (in citricacid) in conjunction with chondroitin sulphate and an oxidising agentcould be used to safely secure a corneal tissue plug to its site ofsurgery.

EXAMPLE 8

[0051] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in 1% lacticacid had its pH adjusted to 7,5-9.5. Chondroitin sulphate (24 mg/ml,aqeous solution) and laboratory chemicals were of highest availablepurity and purchased from Sigma and Merck. The experiments wereperformed on anaesthetised rats according to the rules specified by theethical permissions.

[0052] The cornea was deepithelialized. The surgery and the treatmentwith MAP, chondroitin sulphate and oxidizing agent was performed as inexample 7

[0053] The results obtained agreed with those in example 7 The achievedresults thus indicate that MAP (in lactic acid) in conjunction withchondroitin sulphate and an oxidising agent could be used to safelyreattach a corneal tissue plug to the cornea.

EXAMPLE 9

[0054] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1%lactic acid or in 1% citric acid had its pH adjusted to 7,5-9.5.Chitosan (10 mg/ml, aqeous solution), hyaluronan (10 mg/ml, aqeoussolution) and laboratory chemicals were purchased to be of highestavailable purity. The experiments were performed on anaesthetised ratsaccording to the rules specified by the ethical permissions.

[0055] The cornea was deepithelialized. The surgery and the treatmentwith MAP, hyaluronan or chitosan and oxidizing agent was performed as inexample 7 The results obtained with chitosan roughly agreed with thosein example 7, while hyaluronan worked in the acute situation but no longterm results were achieved. The results indicate that MAP in conjunctionwith additional polysaccharides and an oxidising agent could be used toreattach a corneal tissue plug to the cornea.

EXAMPLE 10

[0056] Purified MAP (0.9 mg/mL, BioPolymer Products AB) in 5% aceticacid had its pH adjusted to become alkaline. Heparin and laboratorychemicals were of highest available purity and purchased from Sigma andMerck. The experiments were performed on anaesthetised rats according tothe rules specified by the ethical permissions.

[0057] The cornea was deepithelialized. Surgery and application of thebonding mixture was performed as in example 5. Adhesion was achievedbetween the central plug and peripheral part of the original cornea, asgently tested after 10 minutes with the aid of ophthalmic tweezers andan operating microscope.

[0058] The results obtained was in good agreement with those presentedin example 5 for in this case MAP in acetic acid and heparin, both withregard to the clinical outcome and the histopatholoy as investigatedafter 5 days.

EXAMPLE 11

[0059] Purified MAP (0.81 mg/ml, BioPolymer Products AB) in 1% lacticacid had its pH adjusted to 7.5-9.5. Heparin and laboratory chemicalswere of highest available purity and purchased from Sigma and Merck.Ophthalmic sutures (10-0) were purchased from Ethicon, cut in smallpieces ranging in length from 1-10 mm and eventually added to the MAPsolution. The experiments were performed on anaesthetised rats accordingto the rules specified by the ethical permissions.

[0060] The cornea was deepithelialised. The cornea was carefully punchedin the centre with aid of a trephine with a diameter of 3 mm. The tissuecylinder was slowly reacted to at least half of its thickness from theoriginal cornea with aid of fine-pointed instruments under microscopicobservation. Two small marks were made in the periphery of the centralcornea plug. A solution, prepared immediately before use, of 20 μl MAPsolution, 10 μl aqueous heparin solution, ophthalmic sutures (10 mg/mlof the final composition)and 10 μl 6% (w/v) aqueous hydrogen peroxidesolution, was applied several times along the border between the plugand the remaining cornea. The cornea plug was then repositioned andgently kept in position. Adhesion was achieved between the central plugand peripheral part of the original cornea after 5-10 minutes, as verygently tested with aid of ophthalmic microsurgery instruments using anoperating microscope.

[0061] Visual inspection and microscopy was performed during thesubsequent 3 hours. There was good adhesion between the central cornealplug and the surrounding residuing cornea. The small defects in theinterface zone were filled by the glue with its filaments.

[0062] The achieved results thus indicate that MAP in conjunction withheparin, an oxidising agent and very fine filaments could be used tosafely glue a corneal plug back to its site of surgery and to fill andbridge defects in the tissue.

EXAMPLE 12

[0063] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1%citric or 1% lactic acid had its pH adjusted to usually to 7,5-8.5, butin some experiments up to about 9.5. Heparin and laboratory chemicalswere of highest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions. The cornea was deepithelialized. Awound was created surgically in the centre through roughly half of thecornea, with the aid of a trephine with diameter of 3 mm. The Bowmansmembrane and stroma was excised with a knife and fine scissors, down tothe vicinity of Descemets membrane. A block of corneal tissue wasthereby isolated and removed from its original site. At least 5 μL ofMAP, 3 μL 10 mg/ml aqueous heparin solution and 2 μL 2 mM sodiumperiodate was administrated into the wound cavity, either once or twice.Thereafter was the shortly before removed corneal tissue piecesrepositioned into the cavity to test for adhesion and reattachmentmediated by the MAP glue. Bonding was achieved between the stromafragments and the wound cavity in the cornea after 5 minutes, as verygently tested with the aid of ophthalmic tweezers and an operatingmicroscope. The MAP-glue combination did not add any obvious extraintensity with regard to the inflammatory reactions during the firstdays. The corneal wound was covered by epithelium within two days. Noadverse effects could be recognised by visual inspection or bymicroscopy that could be related to the MAP composition.

EXAMPLE 13

[0064] Purified MAP (0.81 mg/mL, BioPolymer Products AB) in either 1%citric or 1% lactic acid had its pH adjusted to usually to 7,5-8.5, butin some experiments up to about 9.5. Heparin and laboratory chemicalswere of highest available purity and purchased from Sigma and Merck. Theexperiments were performed on anaesthetised rats according to the rulesspecified by the ethical permissions.

[0065] The cornea was deepithelialized. A wound was created surgicallyin the centre through roughly half of the cornea, with the aid of atrephine with diameter of 3 mm. The Bowmans membrane and stroma wasexcised with a knife and fine scissors, down to the vicinity ofDescemets membrane. A block of corneal tissue was thereby isolated andremoved from its original site. At least 5 μL of MAP, 3 μL 10 mg/mlaqueous heparin solution and 2 μL 2 mM sodium nitroprusside wasadministrated into the wound cavity, either once or twice. Thereafterwas the shortly before removed corneal tissue pieces repositioned intothe cavity to test for adhesion and reattachment mediated by the MAPglue. Bonding was achieved between the stroma fragments and the woundcavity in the cornea after 5 minutes, as very gently tested with the aidof ophthalmic tweezers and an operating microscope. The MAP-gluecombination did not add any obvious extra intensity with regard to theinflammatory reactions during the first days. The corneal wound wascovered by epithelium within two days. No adverse effects could berecognised by visual inspection or by microscopy that could be relatedto the MAP composition.

1. A bioadhesive composition comprising a) a bioadhesive polyphenolicprotein derived from a byssus-forming mussel, which protein comprises30-300 amino acids and consisting essentially of tandemly linked peptiderepeats comprising 3-15 amino acid residues, wherein at least 5% andpreferably 6-25% of the amino acid residues of said bioadhesivepolyphenolic protein are DOPA; b) a polymer comprising carbohydrategroups, such as heparin, chondroitin sulfate, chitosan and hyaluronan;c) pharmaceutically acceptable fine filaments; d) optionally anon-enzymatic oxidising agent such as hydrogen peroxide, nitroprussideions or periodate ions; and optionally e) a filler protein, such ascollagen, casein, albumin, elastin, fibronectin and fibrin; whichcomposition does not comprise any enzymatic oxidising agent or chemicalcross-linking agent.
 2. A bioadhesive composition comprising a) abioadhesive polyphenolic protein derived from a byssus-forming mussel,which protein comprises 30-300 amino acids and consisting essentially oftandemly linked peptide repeats comprising 3-15 amino acid residues,wherein at least 5% and preferably 6-25% of the amino acid residues ofsaid bioadhesive polyphenolic protein are DOPA; b) a polymer comprisingcarbohydrate groups, such as heparin, chondroitin sulfate, chitosan andhyaluronan; c) optionally a non-enzymatic oxidising agent such ashydrogen peroxide, nitroprusside ions or periodate ions; and optionallyd) a filler protein, such as collagen, casein, albumin, elastin,fibronectin and fibrin; which composition does not comprise anyenzymatic oxidising agent or chemical cross-linking agent, for medicaluse.
 3. A bioadhesive composition according to claim 2 furthercomprising pharmaceutically acceptable fine filaments, for medical use.4. Use of a composition according to claim 2 or claim 3 for preparing anophtalmic adhesive which can be used to heal performations, lacerationsor incisions, to reattach the retina to the back of the eye, to repairor attach lenses, and to repair, construct, reconstruct and/or attatchcorneal component parts.
 5. Pharmaceutical compositions containing abioadhesive polyphenolic protein according to claim 1, component a), anda polymer according to claim 1, component b), as a combined bioadhesivepreparation for simultaneous medical use, wherein said combinedbioadhesive preparation does not comprise any enzymatic oxidising agentor chemical cross-linking agent.
 6. Pharmaceutical compositionsaccording to claim 5 further comprising a non-enzymatic oxidising agent,such as hydrogen peroxide, nitroprusside ions or periodate ions. 7.Pharmaceutical compositions according to claim 5 or claim 6 furthercomprising pharmaceutically acceptable fine filaments.
 8. Use ofpharmaceutical compositions according to anyone of claims 1-7, forpreparing a single or combined ophtalmic bioadhesive preparation whichcan be used to heal performations, lacerations or incisions, to reattachthe retina to the back of the eye, to repair or attach lenses, and torepair, construct, reconstruct and/or attatch corneal component parts.9. Use of pharmaceutical compositions according to anyone of claims 1-7,for preparing a single or combined bioadhesive preparation which can beused for treating complications adnexa to the eye, such as facial skinand mucous membranes including eye lids and the conjunctiva, tearchannel system and other periocular structures and the orbit.